Pharmaceutical preparation comprising lyophilized liposomes encapsulating an active principle which is highly insoluble in water, and the process for preparing the said preparation

ABSTRACT

The present invention to relates to a pharmaceutical preparation comprising lyophilized liposomes encapsulating a biologically-active principle which is highly insoluble in water and stable over time.

This application is a 371 of PCT/EP98/00817 filed on Feb. 12, 1998.

The invention relates to a pharmaceutical preparation comprisinglyophilized liposomes encapsulating a biologically-active principlewhich is highly insoluble in water, and a process for preparing the saidpreparation.

More particularly, the invention relates to a pharmaceutical preparationcomprising lyophilized liposomes encapsulating a biologically-activeprinciple which is highly insoluble in water and stable over time.

In the course of the present description and the Claims, the term“highly insoluble in water” is used to describe all those compoundshaving a solubility in water ≦0.01% (w/v).

It is known that the use of liposomes in medical treatments has beenheld back by difficulties encountered in obtaining pharmaceuticalpreparations which are sufficiently stable both during lyophilizationand over time. The said difficulties consist above all in thepreparation of liposomes which neither burst nor pack together. In otherwords, the liposomes should remain whole and separate from one another.

The structural integrity of the liposomes is also particularly importantin the case where the active principle is highly insoluble in water. Inpoint of fact, the bursting of the liposomes during lyophilizationand/or during preservation does not prevent the water-soluble activeprinciples from going into solution when the aqueous liposome solutionis reconstituted before administration to the patient by the addition ofphysiological solution. On the other hand, in the case of the burstingof liposomes comprising active principles which are highly insoluble inwater, reconstitution of the lyophilisate yields a solution comprisingless active principle than required. The greater the quantity of burstliposomes, the greater the difference between the theoretical and theactual quantity of active principle in solution.

A lyophilization method of liposomes comprising a water-solublebiologically-active principle is described by U.S. Pat. No. 4,857,319.This document describes the lyophilization of liposomes preferably of anaverage size of about 50-100 nm, with the addition, as a preservingagent, of a disaccharide on the inside only (with the liposome contentencapsulated), or on the outside only, or on both the inside and theoutside. The disaccharide/lipid weight ratio ranges between 0.1:1 and4:1. Preferably, the disaccharide is trehalose. The freezing phase iscarried out at the temperature of liquid nitrogen (−195.8° C.).

In the aforementioned patent, stability characteristics duringlyophilization were evaluated by means of measurements of retention ofthe active principle encapsulated in the liposomes after reconstitutionof the lyophilisate via rehydration.

Table 2 of the aforementioned patent shows that retention is high(99-100%) only when the trehalose/lipid ratio is greater than 1.76 andthe trehalose is present both on the inside and the outside of theliposomes. When the said ratio is equal to 0.11 and to 0.19, retentionis equal to 22% and 49%, respectively, even if the trehalose is presentboth on the inside and the outside of the liposomes. By contrast, whenthe trehalose is present only on the outside, the amount of activeprinciple retained is drastically reduced, even for very largequantities of trehalose. In point of fact, with a trehalose/lipid ratioof 3.9, the amount retained is only 26%.

Nevertheless, the aforementioned results are not reproducible when thebiologically-active principle is highly insoluble in water. In fact,when the trehalose is added during preparation of the liposomes in orderto encapsulate it within the lipid vesicles, a non-homogeneous,non-extrudible suspension is obtained (Preparations for Comparison 1 and2).

Surprisingly, it has been discovered that liposomes comprising abiologically-active principle which is highly insoluble in water remainsubstantially whole during lyophilization when the trehalose is added insmall quantities to the liposomes before lyophilization only, and thesaid iyophilization is effected by carrying out the freezing phase at atemperature of between −5° and −70° C.

It is an object of the present invention to provide a lyophilizedcomposition comprising trehalose and lipid liposomes in which abiologically-active principle has been incorporated, characterized inthat the biologically-active principle is highly insoluble in water, thetrehalose/lipid weight ratio is ≦1.5, and all of the trehalose was addedto the outside of the liposomes already formed before lyophilization.

After reconstitution by rehydration, the said composition retains insolution more than 95% of the biologically-active principle which ishighly insoluble in water (Examples 1, 2 and 3).

Typical examples of biologically-active principles which are highlyinsoluble in water are: lonidamine, melatonin, cyclosporin A andbindarit.

The lipids of the liposome composition to be subjected to thelyophilization process according to the invention are preferably chosenfrom the group comprising phosphoglycerides, glycerides, diglycerides,triglycerides, phospholipids, galactosyl and glucosyl lipids,cholesterol and its derivatives, sphingolipids and their mixtures.Preferably, the lipids are phospholipids. The trehalose/lipid weightratio, in turn, preferably lies between 1:2 and 1:1.

The average size of the liposomes may be between 50 and 250 nm.Preferably, it is between 50 and 100 nm.

A second object of the invention is constituted by a lyophilizationprocess, characterized in that:

1) from 0.2 to 1.5 parts in weight of trehalose are added for each partin weight of lipids of an aqueous liposome composition in which theaverage size of the liposomes lies between 50 and 250 nm, and the saidliposomes comprise a biologically-active principle which is highlyinsoluble in water;

2) the said composition is chilled via the lyophilizer chilling-plate toa temperature between −5° and −70° C., at a chilling rate of between0.5° and 2° C./min.;

3) once the predetermined freezing temperature is reached, the saidcomposition is kept at the said temperature for a period of between 2and 5 hours;

4) a vacuum of between 5×10⁻¹ and 8×10⁻² millibar is applied, leavingthe temperature of the chilling plate at the chilling temperaturedefined in point 2) for a period lasting between 2 and 5 hours;

5) the temperature of the chilling plate is brought to −1 50° C., andkept there until the water is completely removed.

The preferred operating conditions are as follows:

Phase 2

freezing temperature: −20° to −30° C.

chilling rate: 0.77° C./min.

Phase 3

time: 3 hours

Phase 4

vacuum: 6×10⁻² millibar

Phase 5

a) When the freezing temperature (Phase 2) is below −15° C., thetemperature of the chilling plate is increased to −15° C. at a rate ofbetween 0.5° and 2° C. and lyophilization takes place for 20 hours; thenthe temperature of the chilling plate is brought to −10° C., and afteran hour to +5° C., and lyophilization occurs for 16 hours.

b) When the freezing temperature (Phase 2) is greater than or equal to−15° C., lyophilization is continued for 20 hours, after which thetemperature of the chilling plate is taken to +5° C. and lyophilizationoccurs for 16 hours.

A particularly preferred liposome composition according to the inventioncomprises:

Component % (w/w)

phosphatidylcholine : 94

lysophosphatidylcholine : 3

N-acyl-ethanolamine : 1

phosphatidyl ethanolamine : 0.1

triglycerides : 1

free fatty acids : 0.75

DL-α-tocopherol : 0.15

Typically, the aqueous pharmaceutical liposome composition of theinvention is prepared by:

a) dispersing a biologically-active principle which is highly insolublein water in lipids at a temperature of between 20° and 30° C.;

b) suspending the said dispersion in an aqueous phase;

c) letting the said suspension stand at ambient temperature for a periodof between 0 and 48 hours;

d) heating to between 30° and 75° C. for 10-40 minutes;

e) freezing to between −150° and −2000° C.;

f) repeating phases d) and e) at least twice, and not more than 8 times;

g) filtering through a filtering membrane with pores of 500-1000 nmdiameter;

h) extruding through a membrane with pores of 50-400 nm diameter; andsimultaneous

i) elimination of the active principle not trapped.

The duration of phase c) depends on the quantity of active principlehighly insoluble in water which it is wished to trap in the liposomes.The person skilled in the art will thus have no difficulty indetermining by means of a few simple routine experiments the suitabletime for each type of active principle and liposome composition.

Preferably, the aqueous phase consists of a 0.05%-0.9% (w/v) aqueoussolution of sodium chloride. Typically, the amount of lipids used is0.01-0.4 parts in weight for each part in weight of aqueous solution. Inits turn, the amount of active principle generally lies between 0.01 and0.3 parts in weight for each part in weight of lipids.

Generally, extrusion is effected using as an extrusion gas eithercompressed air or an inert gas chosen from the group comprisingnitrogen, helium and argon. Preferably, helium is the inert gas.Pressure in the extrusion phase is preferably between 500 and 5500 kPa,and the temperature lies preferably between 20° and 750° C., even morepreferably between 40° and 65° C. Typical examples of suitable extrudersare the Lipex Biomembranes Thermobarrel Extruder type, or the EmulsiflexCC Avestin with Costar™ polycarbonate membrane with pores of between 50and 600 nm in diameter.

Proceeding as described above, aqueous liposome compositions areobtained comprising about 8 mg/ml of melatonin, 3.8 mg/ml of lonidamine,1 mg/ml of cyclosporin A and 4 mg/ml of bindarit against awater-solubility of 3×10⁻³ mg/ml (lonidamine), 1×10⁻¹ mg/ml (bindarit)and, practically, the absolute insolubility of melatonin (G. S. Shida etal. “J. Pineal Res.” 1994, 16, 198-201) and of cyclosporin A [“Insolublein Water”, a monograph of cyclosporin A in “Analytical Profiles of DrugSubstances”, 16, 163, (1987)].

The following examples shall serve to illustrate the present invention,without, however, limiting it.

PREPARATION I

A liposome composition comprising a biologically-active principle whichis highly insoluble in water was prepared as described below.

100 mg of melatonin were dispersed in 1 g of phospholipids at 30° C. for10 minutes by means of a Ultraturrax™-type homogenizer. Directlyafterwards, the said dispersion was suspended in 10 ml of 0.9% (w/v)aqueous solution of sodium chloride by means of the said homogenizer andthen heated in a water-bath at 55° C. for 20 minutes.

The suspension thus obtained was subjected to the following chilling andheating cycle:

chilling in liquid nitrogen for 1 minute,

heating at 55° C. until complete fluidization of the phospholipids.

The said cycle was repeated 6 times.

The suspension was passed twice through a 0.6 μm filter using the LipexBiomembranes device.

A “Multilamellar Large Vesicle” (MLV) suspension was thus obtained andsubjected to 6 continuous-extrusion cycles using a 10-mi LipexBiomembranes Thermobarrel type extruder with 0.1 μm Costar™polycarbonate filters at 55° C., using helium as an extrusion gas at apressure of between 1000 and 4800 kPa.

PREPARATION II

We proceeded as described for Preparation I, using 2 g of phospholipidsand 50 mg of lonidamine in place of 1 g of phospholipids and 100 mg ofmelatonin.

PREPARATION III

We proceeded as described for Preparation I, using 2 g of phospholipidsand 200 mg of melatonin in place of 1 g of phospholipids and 100 mg ofmelatonin.

PREPARATION IV

We proceeded as described for Preparation II, except that extrusion wascarried out through a 0.2 μm in place of a 0.1 μm polycarbonatemembrane.

PREPARATION V

30 mg of cyclosporin A were dispersed in 2 g of phospholipids at 30° C.for 10 minutes by means of an Ultraturrax™-type homogenizer. Directlyafterwards, the said dispersion was suspended in a 0.9% (w/v) aqueoussolution of sodium chloride using the said homogenizer, and leftstanding at ambient temperature for 24 hours. Following this, thesuspension obtained was heated in a water bath at 65° C. for 20 minutes.

The suspension thus obtained was subjected to the following chilling andheating cycle:

chilling in liquid nitrogen for 1 minute,

heating at 65° C. until complete fluidization of the phospholipids.

The said cycle was repeated 6 times.

The suspension was passed twice through a 0.6 μm filter using the LipexBiomembranes device.

A “Multilamellar Large Vesicle” (MLV) suspension was thus obtained andsubjected to 6 continuous-extrusion cycles using a 10-ml LipexBiomembranes Thermobarrel type extruder with 0.1 μm Costar™polycarbonate filters at 65° C., using helium as an extrusion gas at apressure of between 1000 and 4800 kPa.

PREPARATION VI

We proceeded as described for Preparation I, using 2 g of phospholipidsand 50 mg of bindarit in place of 1 g of phospholipids and 100 mg ofmelatonin.

PREPARATION FOR COMPARISON 1 Preparation 1A

100 mg of melatonin and 1 g of trehalose were dispersed in 1 g ofphospholipids at 30° C. for 10 minutes by means of an Ultraturrax™-typehomogenizer. Directly afterwards, the said dispersion was suspended in10 ml of 0.9% (w/v) aqueous solution of sodium chloride using the saidhomogenizer, and then heated in a water-bath at 55° C. for 20 minutes.

The suspension thus obtained was subjected to the following chilling andheating cycle:

chilling in liquid nitrogen for 1 minute,

heating at 55° C. until complete fluidization of the phospholipids.

The said cycle was repeated 6 times.

The suspension was passed twice through a 0.6 μm filter using the LipexBiomembranes device.

In this manner, a very dense mass was obtained. The attempt to extrudeit by means of a 10-ml Lipex Biomembranes Thermobarrel type extruderwith 0.1-μm Costar™ polycarbonate filters at 55° C. using helium as anextrusion gas at a pressure of between 1000 and 4800 kPa, wasunsuccessful.

Preparation 1B

We proceeded as described for the preceding Preparation 1A, except thatthe melatonin was omitted. A “Multilamellar Large Vesicle” (MLV)suspension was obtained which turned out to be perfectly extrudible bymeans of a 10-ml Lipex Biomembranes Thermobarrel type extruder with 0.1μm Costar™ polycarbonate filters at 55° C., using helium as an extrusiongas at a pressure of between 1000 and 4800 kPa.

PREPARATION FOR COMPARISON 2 Preparation 2A

We proceeded as described for the purposes of the Preparation forComparison 1A, except that 2 g of phospholipids were used instead of 1.

In this case also, a very dense, non-extrudible mass was obtained.

Preparation 2B

We proceeded as described for the preceding Preparation for Comparison1B, except that 2 g of phospholipids were used instead of 1.

In this case also, a perfectly extrudible MLV suspension was obtained.

EXAMPLE 1

Preparation II was divided up into 1-mi aliquots, and to each aliquotwas added trehalose according to the trehalose/lipid weight ratio givenin Table 1/1.

Lyophilization was carried out in a plate lyophilizer, as follows:

1) chilling to −25° C. at the rate of 0.77° C./min.;

2) maintaining the said temperature (−25° C.) for 3 hours,

3) application of the vacuum (6×10⁻² millibar) and maintenance at thesaid temperature (−25° C.) for 2 hours;

4) heating to −15° C. for 20 hours under a vacuum of 6×10⁻² millibar;

5) heating to −10° C. for 2 hours under a vacuum of 6×10⁻² millibar;

6) heating to +5° C. for 20 hours under a vacuum of 6×10⁻² millibar;

7) locking the vacuum;

8) introduction of air.

The lyophilisate (1 ml) was rehydrated with 1 ml of distilled water andkept at ambient temperature for 30 minutes in order to permit theefficient reconstruction of the liposomes.

0.5 ml of the said solution was further diluted with 10 ml ofphysiological solution in order to measure the average size of theliposomes with the NICOMP 370 device.

Table 1/1 shows the results obtained.

TABLE 1/1 Average size of the liposomes before and after lyophilizationTrehalose/lipids Batch (w/w) Before After LM/302 0 102 911.7 1:2 102115.9 1:1 102 109.7 2:1 102 167.9 LM/303 0 99.2 911.7 1:2 99.2 98.7 1:199.2 109.8 2:1 99.2 291.3 LM/304 0 98.6 911.8 1:2 98.6 94.9 1:1 98.6105.8 2:1 98.6 794

From Table 1/1 we can see that the absence of trehalose entails acertain degree of fusion of the liposomes, evidenced by the increase intheir average size. Surprisingly, the increase in the amount oftrehalose (trehalose/lipids 2:1) also causes a certain degree of fusionwith a consequent increase in the average size.

Similar results have also been obtained with Preparation IV.

The average size of the liposomes and the lonidamine amount weredetermined from a certain number of samples, freshly prepared asdescribed above. Subsequently, the samples were replaced in therefrigerator at 5° C. and sampled at given intervals, rehydrated todetermine the amount of the active principle and the average size of theliposomes. The results thus obtained are given in Table 1/2.

TABLE 1/2 Trehalose/lipid ratio (w/w) 1:2 Time HPLC amount Average sizeBatch (months) (mg/ml) (nm) LM/328 t₀ 3.3 107 1 3.2 110 3 3.2 114 6 3.1127.2 LM/329 t₀ 3.2 103.3 1 3.2 101 3 3.2 110.5 6 3.1 115 LM/330 t₀ 3.399.2 1 3.4 99.5 3 3.2 100.5 6 3.1 113.6

EXAMPLE 2

Preparation III was lyophilized as described in Example 1 above, and theaverage size of the liposomes before and after lyophilization (Table2/1), as well as the average size of the liposomes and the amount ofmelatonin in the fresh preparations and in those kept at 5° C., weredetermined as described in the aforementioned example (Table 2/2).

TABLE 2/1 Average size of the liposomes before and after lyophilizationTrehalose/lipids Batch (w/w) Before After LM/336 0 92 16953 1:2 92 68751:1 92 96.5 2:1 92 109.7 LM/337 1:2 92 146.3 1:1 92 98.8 2:1 92 8319.8LM/338 1:2 92 179.7 1:1 92 225.7 2:1 92 2984

Similar results were also obtained with Preparation 1.

TABLE 2/2 Trehalose/lipid ratio (w/w) 1:1 Time HPLC amount Average sizeBatch (months) (mg/ml) (nm) LM/359 t₀ 6   104 1 6   103.2 3 5.8 109.5LM/360 t₀ 6.5 107.2 1 6.5 106.3 3 6.7 113.6 LM/361 t₀ 6.2 98.4 1 6.3 993 6   100.5

EXAMPLE 3

Preparation VI was lyophilized as described in Example 1 above, and theaverage size of the liposomes before and after lyophilization (Table3/1) as well as the average size of the liposomes and the amount of thebindarit in the fresh preparations and in those kept at 5° C., weredetermined as described in the aforementioned Example (Table 3/2).

TABLE 3/1 Average size of the liposomes before and after lyophilizationTrehalose/lipids Batch (w/w) Before After LM/342 0 102.7 7524.1 1:2102.7 928 1:1 102.7 109 2:1 102.7 140 LM/343 1:2 102.7 546.6 1:1 102.7112.3 2:1 102.7 1559.2 LM/344 1:2 102.7 194.9 1:1 102.7 112.2 2:1 102.74722

TABLE 3/2 Trehalose/lipid ratio (w/w) 1:1 Time HPLC amount Average sizeBatch (months) (mg/ml) (nm) LM/356 t₀ 3.2 135.4 1 3.1 128 3 3.2 131.3LM/357 t₀ 3.1 122 1 3.1 121 3 3.2 126.2 LM/358 t₀ 3.1 126 1 3.1 122.8 32.9 121.8

EXAMPLE FOR COMPARISON 1

Preparation II was divided into 1 ml aliquots and trehalose was added toeach aliquot according to the trehalose/lipid weight ratio given inComparison Table 1.

The Preparation was then frozen at the temperature of liquid nitrogen(−195.8° C.) and lyophilized for 20 hours, with no external temperaturecontrol.

lyophilized (1 ml) was rehydrated with 1 ml of distilled water and keptat ambient temperature for 2 hours.

0.5 ml of the said solution was further diluted with 10 ml ofphysiological solution in order to measure the average size of theliposomes with the NICOMP 370 device.

The results obtained are illustrated in Comparison Table 1 below.

COMPARISON TABLE 1 Average size of the liposomes before and afterlyophilization Trehalose/lipids Batch (w/w) Before After LM/302 0 102   911.8 1:2 102    254.8 1:1 102    219.3 2:1 102    773.7 LM/303 0 99.2911.8 1:2 99.2 349.9 1:1 99.2 180.4 2:1 99.2 717.2 LM/304 0 98.6 911.81:2 98.6 722.5 1:1 98.6 161.1 2:1 98.6 150  

From Comparison Table 1 we can observe that when lyophilization iscarried out at the temperature of liquid nitrogen, either in the absenceof trehalose or in the presence of trehalose in the ratios given in theTable above, a certain degree of fusion of the liposomes takes place,evidenced by the increase in their average size. In addition, the abovedata indicate that when lyophilization is carried out at the temperatureof liquid nitrogen, the course of the lyophilization itself (forpreparations of the same composition) does not always reproduce the sameresults.

What is claimed is:
 1. Lyophilized composition comprising trehalose and lipid liposomes in which a biologically-active principle has been incorporated, wherein the biologically-active principle is highly insoluble in water, the trehalose/lipid weight ratio is between 2:1 and 1:2, and all of the trehalose was added to the outside of the liposomes already formed before lyophilization.
 2. Lyophilized composition according to claim 1, wherein the biologically-active principle is selected from the group consisting of lonidamine, melatonin, cyclosporin A and bindarit.
 3. Lyophilized composition according to claim 1, wherein the lipids arc selected from the group consisting of phosphoglycerides, glycerides, diglycerides, triglycerides, phospholipids, galactosyl lipids, glucosyl lipids, cholesterol, cholesterol derivatives, sphingolipids and their mixtures.
 4. Lyophilized composition according to claim 3, wherein the lipids are phospholipids.
 5. Lyophilized composition according to claim 1, wherein the trehalose/lipid weight ratio is between 1:2 and 1:1.
 6. Lyophilized composition according to claim 1, wherein the average size of the liposomes is between 50 and 250 nm.
 7. Lyophilized composition according to claim 6, wherein the average size of the liposomes is between 50 and 100 nm.
 8. Process for lyophilizing a composition comprising trehalose and lipid liposomes, in which there has been incorporated a biologically-active principle: a) adding trehalose to the liposome wherein the ratio of the trehalose to liposome is between 1:2 and 2:1, wherein the average size of the liposomes is between 50 and 250 nm, the said liposomes comprising a biologically-active principle which is highly insoluble in water; b) the said composition is chilled by means of the chilling plate of the lyophilizer to a temperature between −5° and −70° C., at a chilling rate of between 0.5° and 2° C./min.; c) once the predetermined freezing temperature has been reached, the said composition is kept at the said temperature for between 2 and 5 hours; d) a vacuum of between 5×10⁻¹ and 8×10⁻² millibar is applied, leaving the temperature of the chilling plate at the chilling temperature defined in point b) for a period of between 2 and 5 hours; e) the temperature of the chilling plate is brought to −15° C., and kept there until the water is completely removed.
 9. Lyophilization process according to claim 8, wherein, in Phase b), the freezing temperature is between −20° and −30° C.
 10. Lyophilization process according to claim 8, wherein, in Phase b), the chilling rate is 0.77° C./min.
 11. Lyophilization process according to claim 8, wherein, in Phase c), the time is 3 hours.
 12. Lyophilization process according to claim 8, wherein, in Phase d), the vacuum is 6×10⁻² millibar. 